K. J. Kasha

An improved in vitro technique for isolated microspore culture of barley

A detailed procedure for isolated microspore culture of barley is presented along with examples of response across genotypes. Over 30 genotypes, including winter and spring growth habit and 2-row and 6-row genotypes, have shown an essentially genotype independent response, averaging about 10,000 embryos per 5 cm petri culture plate. The regeneration frequency, checked on samples of 500 embryos per plate ranged from 36 to 97% with most genotypes being in the range of 70 to 90%. About 70 to 80% of the plants regenerated have been completely fertile doubled haploids, thus eliminating the need to double the chromosome number of plants. Many little details are critical to success of the microspore procedure and while it saves much time compared to anther culture, greater attention to details and cleanliness is essential.

Haploids in Cereal Improvement: Anther and Microspore Culture

For years, cereals and monocots in general have been poor responders in in vitro haploid production systems when compared to many dicot crops. The response frequency was low with very strong genotype effects and often large numbers of albino progeny. However, we are now seeing evidence of a breakthrough that can provide embryo frequencies equivalent to those obtained in some Brassicas and other dicot crops. These results are with specific genotypes of barley (Hordeum vulgare L.) (Hunter, 1988; Hunter et al., 1989; Olsen, 1987), where green plant production has increased up to 100 fold. As a result, further improvements in wheat (Triticum aestivum L.) and other cereals will likely follow. In addition, plants have been obtained from isolated microspore culture in barley, wheat, rice (Oryza sativa L.) and corn (Zea mays L.). These cultures open up new possibilities for transformation, mutation and selection in cereals.

Arabinogalactans and arabinogalactan-proteins induce embryogenesis in wheat (Triticum aestivum L.) microspore culture

The objective of this study was to improve induction of embryogenesis in wheat microspore culture in order to obtain a high number of regenerable embryos. The arabinogalactan (AG) Larcoll and the arabinogalactan-protein (AGP) from gum arabic were tested on two spring genotypes to see if they could increase microspore viability and induce embryogenesis in the microspore culture. Adding Larcoll significantly decreased microspore mortality in both genotypes regardless of the presence or absence of ovaries in the culture. Similarly, gum arabic had a strong effect on the number of embryos produced and regenerated green plants. In fact, by using only gum arabic we were able to obtain green plants from wheat microspore cultures without the presence of ovaries. In addition to preventing a high mortality rate of the cells, our results show that the induction of embryogenesis in wheat microspore cultures is strongly affected by the use of both AG or AGP.

Ethylene production and embyogenesis from anther cultures of barley (Hordeum vulgare).

SummaryEthylene production was measured in cultured barley (Hordeum vulgare L.) anthers. The pattern of ethylene production and the content of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were different among cultivars. Ethylene production appeared to be related to embryogenesis (callus and embryo production). In cultivars in which anthers had low amounts of ACC and produced ethylene slowly, the addition of ethylene promotors (Ethrel or ACC) increased embryogenesis. However, in the cultivar Klages, in which anthers had high amounts of ACC and produced ethylene rapidly, the addition of an ethylene production inhibitor (putrescine) increased embryogenesis. Thus, an optimum level of ethylene production appears to be important for embryogenesis. The differences in anther response and callus production among cultivars may be due to both the capacity to produce ethylene and the sensitivity to high ethylene levels.

Improved plant regeneration from wheat anther and barley microspore culture using phenylacetic acid (PAA).

SummaryThe effect of the auxin phenylacetic acid (PAA) on wheat anther and on barley anther/microspore culture was investigated. With PAA the induction response was not usually significantly different from controls but a significantly higher number of green plants were produced in wheat anther and barley microspore culture. For wheat anther culture 100 mg/L PAA was beneficial. For barley microspore culture the optimum levels were from 1 to 100 mg/L, depending on genotype. In barley anther culture there were no improvements using PAA. In wheat anther culture, 145 green plants/100 anthers were obtained with cultivar Veery‘S’, while the average response from twelve F1 hybrids in the breeding program was 332 green plants/100 anthers. At least 1000 green plants were obtained using isolated microspores from 100 anthers in barley cv. Igri. With cv. Bruce, regeneration occurred only when 100 mg/L PAA was used. The influence of PAA appears at the embryogenic phase of the culture system. The possible mechanisms by which PAA may improve regeneration are discussed.

Published doubled haploid protocols in plant species

Forty-four protocols of doubled haploid production (DH) are presented in this Manual. They are related to at least 33 plant species, not counting protocols on production of DH from interspecific hybrids. The protocols provide procedures for production of DH in such major crops as wheat, maize, barley, rapeseed or potato and also in other crop species where this technology is more advanced, or in other words, where a relatively high frequency of doubled haploids was obtained. However, there have been several approaches to develop doubled haploids in many crops or plant species. We are presenting below the list of publications related to DH production in 226 additional plant species. This list does not include protocols of plant species published in Chapter 2 of this Manual. This means that efforts have been undertaken to develop doubled haploids in more than 250 plant species as the presented list was built on the basis of our own collection of related publications and as a result of searching in publicly available databases. It was impossible to cite all published information. We limited references to those which carried protocols or more recent information on DH production. In the cited papers, presented protocols did not always lead to the regeneration of haploid or doubled haploid plants. However, even negative results can be very helpful for someone who is initiating this work. We hope that publication of this list will help in further application of DH technology in crop improvement and basic research.

Improved plant regeneration from shed microspore culture in barley (Hordeum vulgare L.) cv. Igri

This report describes rapid regeneration of green plants from microspores of the barley cultivar Igri. Use of 0.3 M mannitol during maceration and isolation was essential for response from mechanically isolated microspores of barley cv. Igri grown under our conditions. A shed microspore culture system proved to be simple and gave a fast response; plants were obtained as early as 25 days after the material was taken from the donor plant. A 28-day cold-pretreatment of spikes can also be replaced with a 3–4 day pretreatment of anthers in mannitol. Shed microspores from 100 anthers produced an average of 292 plants with 91% of them green. Approximately 80% of the regenerated plants were spontaneously doubled-haploids.

Visual screening of microspore-derived transgenic barley (Hordeum vulgare L.) with green-fluorescent protein

Abstract. The green-fluorescent protein (GFP) gene from the Pacific Northwest jellyfish, Aequorea victoria, was used as a screenable marker in the production of transgenic barley plants. Isolated barley microspore culture was biolistically transformed with two synthetic forms of GFP, sgfp and pgfp. Thirty-seven fluorescing multicellular structures were isolated using epifluorescent microscopy. Sixteen structures developed shoots, but only five regenerated into green plants. Three events had been co-bombarded with β-glucuronidase (gus) and assayed positive for gus expression in the leaves, and all five events were positive for gfp expression. The expected transgene band size was PCR-amplified from all five plants, and Southern blots performed on three plants revealed unique patterns of gfp transgene integration. Fluorescent in situ hybridization also revealed the transgenic status and hemizygous nature of all the events. GFP-based visual screening provides a viable alternative method to chemical selection of transgenic plants from barley microspore culture.

ISOLATED MICROSPORE CULTURE OF WHEAT (TRITICUM AESTIVUM L.) IN A DEFINED MEDIA I. EFFECTS OF PRETREATMENT, ISOLATION METHODS, AND HORMONES

Significant improvements were achieved in the production of haploid and doubled haploid plants from isolated microspore culture of wheat c.v. Chris on a defined media. Procedures found to be of benefit included: A 7-day pretreatment of anthers in 0.4M mannitol plus the macronutrients from FHG medium; the inclusion of 4.5 mg/liter abscisic acid in the pretreatment solution; the isolation of microspores from pretreated anthers by vortexing; and the use of phenylacetic acid (PAA) as the auxin source in MS medium. The best response was achieved with 4.0 mg/liter PAA in MS medium containing 90 g/liter maltose as the sugar source. Under these conditions, 68% of viable microspores underwent division, and an average of 93 embryos and 92 green plants were regenerated per 100 anthers used. The root-tip chromosome number and the fertility of 114 regenerating green plants revealed that 75% were completely fertile spontaneously doubled haploids.

Direct DNA uptake during the imbibition of dry cells

Abstract The feasibility of inserting DNA into dry plant cells during imbibition was investigated using dry embryos of alfalfa (Medicago sativa L.). Somatic embryos produced in vitro from cell culture, were induced to become desiccation tolerant and dried to approx. 10–15% moisture. In the dry state, the lipid bilayer of the plasmalemma is disorganized and is increasingly permeable to cytoplasmic solutes. To determine whether it was also permeable to large plasmid DNA molecules, dry somatic embryos were imbibed in a solution containing a plasmid carrying the β-glucuronidase (GUS) reporter gene. Transient expression of the GUS gene was observed visually in germinating embryos and seedlings, suggesting that DNA uptake can occur during the imbibition of dry cells. This simple, direct DNA uptake method may be used for inserting foreign DNA into plant cells for both transient and stable genetic transformation.